Hi everyone,
I have the following situation:
We want to clone a ~1.4bp construct into a pGEX-4T plasmid for protein production. For this, we ordered a gene fragment (verified by mass spec) and since the fragment had restriction sites flanking it, we went for PCR (FIRE polymerase) and classical cloning into the backbone instead of Gibson assembly, partly because of the varying efficiency of our competent cells.
After getting one colony, the sequencing data (checked in fwd and rev direction by now) show a 27bp gap. I don't know where i can expect the gap to come from. Did something like that happen to you in the past? I think the 2 possibilities are that the polymerase skipped it, although there were no repetetive elements, predicted and a GC-content of ~50%, or the base pairs were missing in the first place which was not caught by the mass spec analysis.
Further, do you have a suggestion for a quick fix for the construct we already have now? Maybe someone had success with some overhang-PCR approaches in a similar scenario.
Thanks in advance for your help!
Dear Rob
you can easly insert a 28bp fragments using PIPE cloning . You need just to desing 2 primers to perform the vector PCR with contains overlapping flag regions, perform the pcr of the entire vector (0,1ng template/25ul reaction volume) using an high fidelity polimerase, perform dpni digestion and trasfrom 2ul of it in mach1 competent cells (thermo). Generally if you see a good PCR band, the most of colonies contains the right clones.
It is very simple and fast (you do not need any PCR purification steps). I used it several times to introduce leader sequences (as pelB, OmpA) or other additional sequences (eh His tag, kozac sequence, TEV digestion site) in many clones suitable for recombinant protein expression.
You need to carefully desing the primers but once you have it, in 2-3 days you can send the colonies to sequence. Of course it work well with standard vectors (5-7 Kb) while can be more challenging for larger vectors, becuase not all the polimesares are able to amplify it.
You can find more information about PIPE cloning and primer desing for PIPE cloning on my Blog: ProteoCool ( http://proteocool.blogspot.com/ )
good luck
Manuele
Before trying to fix the defective clone, I would try to sequence a few (2-3) more clones. If those also turn out to have spurious mutations, I would start over with a new PCR reaction.
UPDATE:
Good thing that we already let the plates grow a bit longer: a tiny colony we couldn't see before seems to contain the whole construct from the gap, so it seems our FIRE-based PCR skipped the 27 bp in some cases.
Therefore, we'll just confirm the rest of the sequence by sequencing and contionue with the project.
Thank all of you so much for your recomendations!
Have a wonderful weekend,
Rob
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